Color-developing Reagent Research Articles

Screening of natural inhibitors against peptidyl arginine deiminase 4 from herbal extracts by a high-performance liquid chromatography ultraviolet-visible based method

Peptidyl arginine deiminase 4 (PAD4) is an important biocatalytic enzymes involved in the conversion of protein arginine to citrulline, its dysregulation has a great impact on many physiological processes. Recently, PAD4 has emerged as a potential therapeutic target for the treatment of various diseases including rheumatoid arthritis (RA). Traditional Chinese Medicines (TCMs), also known as herbal plants, have gained great attention by the scientific community due to their good therapeutic performance and far fewer side effects observed in the clinical treatment. However, limited researches have been reported to screen natural PAD4 inhibitors from herbal plants. The color developing reagent (COLDER) or fluorescence based methods have been widely used in PAD4 activity assay and inhibitor screening. However, both methods measure the overall absorbance or fluorescence in the reaction solution, which are easy to be affected by the background interference due to colorful extracts from herbal plants. In this study, a simple, and robust high-performance liquid chromatography ultraviolet-visible (HPLC-UV) based method was developed to determine PAD4 activity. The proposed strategy was established based on COLDER principle, while used hydrophilic l-arginine instead of hydrophobic N-benzoyl-l-arginine ethyl ester (BAEE) as a new substrate to determine PAD4 inhibition activity of herbal extracts. The herbal extracts and PAD4 generated hydrophobic l-citrulline were successfully separated by the HPLC, and the developed method was optimized and validated with a known PAD4 inhibitor (GSK484) in comparison with COLDER assay. The IC50 value of GSK484 measured by HPLC-UV method was 153 nM, and the detection limit of the citrulline was 0.5 nmol, respectively, with a linear range of 0.5 nmol to 20 nmol. The IC50 value of the HPLC-UV method was improved by nearly three times compared with COLDER assay (527 nM), and the results indicated the reliability of PAD4 inhibition via HPLC-UV method. The inhibitory effect against PAD4 were fast and accurately screened for the twenty-four extracts from eight herbs. Among them, Ephedra Herba extracts showed significant inhibitory activity against the PAD4 with the IC50 values of three extracts (ethanol, ethyl acetate and water) ranging from 29.11 μg/mL to 41.36 μg/mL, which may help researchers to discover novel natural compounds holding high PAD4 inhibition activity.

Optimization of dissolved urea measurements in coastal waters with the combination of a single reagent and high temperature

Urea is an unstable and intermediate organic nitrogenous compound present in coastal environments and is derived from the excretion of some aquatic organisms and wastewater discharges. Urea plays an important role in the nitrogen cycle, where it is utilized by algae, including diatoms. However, there are very limited relevant data on the production, consumption, and degradation of urea because of the lack of appropriate measurement techniques. The conventional method is based on the formation of a colored product when urea reacts with diacetyl monoxime in a sulfuric acid solution. We examined the optimal conditions for the formation of the colored product; specifically, we evaluated different temperatures (22–80 °C), reaction times, mixing ratios of color reagents, and sample storage times. Application of the single mixed color-developing reagent (COLDER) at 70 °C resulted in the optimal formation of the colored product within a short reaction time of 60 min. This method was then used to measure dissolved urea in different coastal environments. The concentrations detected were as follows: 0.65–0.72, 0.49–0.58, and 1.09–2.28 µM urea-N at coral reef, seagrass, and mangrove sites, respectively. Our results showed high precision (SD = 0.02, CV = 1.2%), a low detection limit (0.03 µM urea-N), and a high recovery rate (94–99%). In summary, this high-temperature procedure for urea measurements should be valuable for obtaining high-precision data that can further the understanding of urea dynamics and its role in coastal ecosystems.

An efficient colorimetric assay for RNA synthesis by viral RNA-dependent RNA polymerases, using thermostable pyrophosphatase

RNA-dependent RNA polymerase (RdRp) is essential for the replication of RNA genome-containing positive-strand RNA viruses. We developed a simple colorimetric assay to quantify the RNA synthesis activity of RdRp by measuring the pyrophosphates released during nascent RNA synthesis. RNA polymerase reaction was quenched by heating at 70°C for 5min, during which thermostable inorganic pyrophosphatase converted the accumulated pyrophosphates into inorganic phosphates. Subsequently, the amount of inorganic phosphate was measured using a color-developing reagent. Using RdRp’s from hepatitis C virus and foot-and-mouth disease virus, we demonstrate that this colorimetric assay facilitates the measurement of RNA polymerase activity.

Colorimetric Grading Scale Can Promote the Standardization of Experiential and Sensory Evaluation in Quality Control of Traditional Chinese Medicines

Experiential and sensory evaluation is an ancient method that remains important in the current quality control system of Traditional Chinese Medicines (TCMs). The process is rapid and convenient when evaluating the quality of crude materials in TCM markets. However, sensory evaluation has been met with skepticism because it is mainly based on experience and lacks a scientific basis. In this study, rhubarb was selected to demonstrate how color-based sensory evaluation could differentiate the quality of herbal medicines objectively. The colors of the rhubarb samples, expressed as RGB values, were obtained from different parts and forms of the plant, including the plant’s surface, fracture surface color, and a powdered form with or without treatment with a color-developing reagent. We first divided the rhubarb samples into three grades based on the total content of five hydroxyanthraquinone derivatives, the major pharmacological components in rhubarb. Then, a three-layer back-propagation artificial neural network (BP-ANN), calibrated with selected training samples, was used to correlate the quality of the rhubarb with its color. The color of the rhubarb powder after coloration attained the highest accuracy (92.3%) in predicting the quality grade of the test samples with the established artificial neural networks. Finally, a standardized colorimetric grading scale was created based on the spatial distribution of the rhubarb samples in a two-dimensional chromaticity diagram according to the colors of the powdered rhubarb after color enhancement. By comparing the color between the scale and the tested samples, similar to performing a pH test with indicator paper, subjects without sensory evaluation experience could quickly determine the quality grade of rhubarb. This work illustrates the technical feasibility of the color-based grading of rhubarb quality and offers references for quantifying and standardizing the sensory evaluation of TCMs, foods and other products.

Efficient Colorimetric Assay of RNA Polymerase Activity Using Inorganic Pyrophosphatase and Ammonium Molybdate

3Therefore, continuous hydrolysis of pyrophosphate by pyrophophatases can enhance the yield of RNA or DNA synthesis during the nucleic acid polymerization. We have set out the experiments to monitor the activity of nucleic acids polymerase using the PPase, which can be applicable to a high-throughput screening of antiviral drugs against various viral RNA polymerases. Currently, activity assay of RNA polymerases is mainly based on the direct measurement of RNA polymerase’s incorporation of radioactive ribonucleotides in the synthesizing nucleic acids. Such radioactive assays cannot be easily adapted to high throughput screening. In our studies, the amounts of inorganic phosphate are to be quantitatively measured with color-developing reagent after the RNA polymerase reaction, which is generated from hydrolysis of pyrophosphates by the PPase. This colorimetric coupledenzyme system will facilitate measurement of RNA polymerase activity without need of cumbersome radioactivity assay.

High performance liquid chromatographic determination of acetoacetate by post-column derivatization with p-nitrobenzene diazonium fluoroborate.

We have applied a color-developing reagent, p-nitrobenzene diazonium fluoroborate (diazo reagent) as a post-column derivatization tool for the specific determination of acetoacetate (AcAc) in high performance liquid chromatography (HPLC). A mobile phase consisting of 50 mM KH(2)PO(4), 4 mM tetra-n-butylammonium phosphate (TBAP) as an ion-pair reagent and 2 v/v% methanol, pH 3.5, diazo reagent solution with 0.2% triton X-100, and alkaline solution of 1.5 mol/l NaOH were pumped using three independent pumps. Specific color development on-line was monitored at 645 nm. A calibration curve for AcAc standard solution with an injection volume of 20 microl showed a good linearity in the range 0.01-2.5 mM with a correlation coefficient of 0.999. For the determination of 3-hydroxybutyrate (3-HOBA), 3-HOBA was converted to AcAc by an enzymatic-coupling method using 3-HOBA dehydrogenase and lactate dehydrogenase. Analytical recoveries of AcAc and 3-HOBA added to serum and urine were satisfactory.

Synthesis of a new color-developing reagent for the spectrophotometric determination of soaps on the basis of an ion-association reaction in aqueous solution.

Synthesis of a new color-developing reagent for the spectrophotometric determination of soaps on the basis of an ion-association reaction in aqueous solution.

Determination of Soluble Phosphorus in the Presence of Organic Ligands or Fluoride

AbstractIn the ascorbic acid‐molybdenum blue method for measurement of soluble P, the relationship between color intensity and P concentration in solution and color stability is greatly affected by organic and inorganic ligands such as oxalate, citrate, tartrate, or F. The objective of this study was to evaluate the effect of these ligands and to optimize the conditions for determination of inorganic P in the presence of the interfering ligand. The critical concentrations of the ligand at which P recovery was significantly decreased were 1.5 mM for oxalate, 3.0 mM for citrate or tartrate, and 10 mM for fluoride. The interference of the ligand with P determination was overcome by excess amounts of ammonium molybdate (AM) added before the color developing reagent (CDR). The critical molybdate/ligand molar ratios (excluding the amount of molybdate added in the CDR) required to completely eliminate the interference of the ligand were 1.12 for oxalate, 0.84 for citrate, 0.56 for fluoride, and 0.34 for tartrate. The presence of ligand and excess amount of AM did not affect the linearity of color intensity against P concentration but did alter the slope, suggesting that the modified method is reliable for P determination in the presence of an interfering ligand provided that the standard curve is prepared in the same matrix as the sample.

An automatic photometric method with high precision by gradually diluting a sample

An automatic photometric method to increase the precision of measurement is proposed, which is based on the injection of a very small volume of sample by rotary valve and the subsequent homogeneous mixing in a small reaction cell made of glass of the sample with a color-developing reagent gradually delivered by a syringe piston pump until the absorbance of the mixture approaches 0.434, where the lowest relative error in the concentration locates. This photometric method is characteristic of uniformly high precision, low sample and reagent consumption, and high sampling rate of 30 samples per h. The other advantages of this method include easy calibration with only one standard solution and no strict requirements on photometric components. While applied for the determination of cobalt ranging from 0.21 to 1.43 g 1 −1, the relative standard deviations are all below 0.4%.

EFFECTS OF ACIDITY AND HYDROXYLAMINE ON THE DETERMINATION OF ALUMINUM WITH FERRON

Ferron (8-hydroxy-7-iodo-5-quinoline sulfonic acid) is a dibasic acid (H2A) which dissociates to HA- and A2- with increased pH. The results show that only HA-, which is the predominant species around pH 5, is reactive with Al. The Al-ferron complex yields an absorption maximum at 366 nm, with a molar absorptivity of 7,940. H2A and A2- are not reactive with Al, while they both yield a strong absorption background near 366 nm. The variation in the distribution of various ferron species in solution accounts for the acidity effect on Al determination. Based on the above information, the color-developing reagent was prepared by mixing ferron, Na acetate, and acetic acid together. This recommended reagent should have pH 5.2. With the addition of 5 mmol HCl to a sample aliquot in Al determination, the final color solution (50 mL) should have pH 4.75 when the solution is still dominated by HA-. The solution also has a high buffer capacity to tolerate the variation in acidity. The addition of hydroxylamine gradually alters ferron to a different chemical species. This hydroxylamine-modified species also reacts with Al and yields an absorption maximum at 362 nm, with a molar absorptivity of 10,500. Because of the changes in absorption maximum and molar absorptivity with time, it is necessary to age the reagent for at least 5 days before using. By deleting hydroxylamine in preparation, the ferron reagent can be used immediately after preparation.

Study on the color reaction mechanism of the Seliwanoff reaction with 4-ethylresorcinol as the color-developing reagent.

4-Ethylresorcinol was found to be a very sensitive color-developing reagent for the detection of ketoses, and was used to elucidate the reaction mechanism of the Seliwanoff reaction. The reaction product (I) responsible for the characteristic coloration of the Seliwanoff reaction was isolated and determined to be 2, 7-diethyl-6-hydroxy-9-(5-methyl-2-furyl)-3H-xanthen-3-one. Compound I was also isolated as crystals from the reaction mixture obtained by Kulka's modified method using 4-ethylresorcinol as the color-developing reagent.

A sensitive method for the determination of free fatty acids in plasma.

A sensitive micromethod for the colorimetric determination of free fatty acids (FFA) in plasma has been developed. FFA are extracted with chloroform-heptane-methanol with silicic acid being added to eliminate interfering phospholipids. Diphenylcarbazide containing diphenylcarbazone has been chosen as color-developing reagent, since the chromophore formed in the presence of FFA-Cu complex is stable and sensitive. Comparison of the proposed method and a non-routine reference procedure involving, among other steps, isolation of FFA on thin-layer chromatography would indicate that the efficiency of extraction of FFA in the proposed method was 90% that of the reference procedure. The proposed method offers the sensitivity, convenience, and accuracy needed for the determination of FFA in situations where the size of sample available is limiting, as with small rodents.

Colorimetric determination of nitrate after hydrazine reduction to nitrite

Since nitrate could be a precarcinogen or a pretoxicant, several sensitive, reproducible manual methods for the determination of nitrate based on the coppercatalyzed reduction of nitrate to nitrite with hydrazine sulfate have been developed. Optimum conditions were obtained using sulfanilamide and N-(1-naphthyl)ethyl-enediamine dihydrochloride as the color-developing reagent. Two new and even more sensitive pairs of reagents for nitrate analysis are introduced that do not involve the use of a possibly carcinogenic naphthylamine. These were 4-amino-acetophenone and azulene or 8-anilino-1-naphthalenesulfonic acid.

An improved method for the colorimetric determination of free fatty acids

An improved method is proposed for the colorimetric microdetermination of free fatty acids in biological fluids, especially in icteric sera. The previous method could not be applied to icteric sera, because some pigment which had passed into the chloroform phase interfered with colorimetry at 440 mμ. Therefore, the following two improvements were introduced. 1. 1. Omission of the pigment from the final colored phase which is to be measured. 2. 2. Alteration of the wavelength of colorimetry from 440 mμ to 600 mμ by using bicyclohexanoneoxalyldihydrazone instead of diethyldithiocarbamate for the color-developing reagent. The sensitivity was intensified by more than 20% compared with that of the previous method.

Thiothenoyltrifluoroacetone: a new chelon

The thioderivative of 2-thenoyltrifluoroacetone, 1,1,1-trifluoro-4-(2-thienyl) 4-mercaptobut-3-en-2-one (STTA) was prepared. NMR and infrared spectra confirmed the previously suggested structure2. The sulfur is attached to the carbon adjacent to the thienyl group and exists almost entirely in the thio-enolic form. The chelates of STTA with Ni(II), Cu(II), Pb(II), Zn(II). Co(II), Cd(II). Pd(II) and Hg(II) were prepared in a neutral to slightly basic medium, and characterized by analysis. NMR, infrared, visible and ultraviolet spectra, DTA, TGA and fractional sublimation. The chelates are relatively stable, insoluble in water, soluble in non-polar and some polar organic solvents, sublimable, and intensely colored compared to their normal diketone analogs. The selectivity of the reagent has not been completely established but divalent ions chelate more readily than trivalent ions; Fe(III), Al(III), Cr(III) apparently do not chelate. The new chelon may be useful as a color-developing reagent for the colorimetric analysis of such ions as Zn(II), Cd(II), Hg(II) and Pb(II). The molar extinction coefficients are given for the chelon and the chelates investigated

Tetracyanoethylene as Color-Developing Reagent for Aromatic Hydrocarbons

Tetracyanoethylene as Color-Developing Reagent for Aromatic Hydrocarbons

Spectrophotometric Determination of Iron in Serum

Abstract To improve the procedure of photometric determination of iron in human serum, Torii’s method recently proposed for the determination of iron was applied to serum. After the removal of protein from serum, the iron in serum is reduced, the pH of the solution is adjusted to 4.5, the color-developing reagent is added, and the excess of the reagent is removed by extraction with carbon tetrachloride. The green solution thus obtained is effective for spectrophotometric determination. The interference of copper is avoided by the addition of thiosulfate, and the substances other than copper remaining after the removal of protein from serum do not give any appreciable interference.

A Simple Modified Method for the Determination of Vitamin A in Sterile Milk

Summary Activated glycerol dichlorohydrin was used as the color-developing reagent instead of antimony trichloride. Saponification and extraction of unsaponifiable procedure was omitted since the fat was directly used after dilution. The time required is less than the method for extracting the unsaponifiable fraction.